7. PROJECT SUMMARY/ABSRACT Methicillin-resistant Staphylococcus aureus (MRSA) is among the most frequently reported isolates of healthcare-associated infections. Although increased prevention efforts in the United States have led to a recent decline in these infections, MRSA will remain a formable threat to public health. Accordingly, there is a continual need to develop new treatment strategies to maintain a sufficient number of therapeutic options for clinicians to treat vancomycin-susceptible (VSSA) and less common vancomycin-intermediate resistant (VISA) infections. In addressing the need to develop new therapeutic strategies, we will investigate the use of disulfiram (Antabuse?) as an antibiotic adjuvant in the treatment of MRSA infections with reduced vancomycin (VAN) susceptibility. Our preliminary research has established that disulfiram (DSF), an oral prescription medication for alcohol abuse disorder, decreases the minimum inhibitory concentration (MIC) of VAN in VISA (4-8 g/mL) to MIC levels observed for VSSA (? 2 g/mL). This project will conduct a more detailed analysis of the ability of DSF to alter VSSA and VISA susceptibility to VAN through the differential analyses of MIC90/MBC90 values, time-kill rates, and postantibiotic effects. Meanwhile, our research team will use whole- genome sequencing, RNA-seq, and HPLC thiol metabolomics to decipher the mechanisms of DSF growth inhibition in S. aureus. The findings of those studies will be qualified by phenotypic screening of S. aureus transposon mutants and homologous recombination experiments. The final aim of the investigation will test our hypothesis that parenteral VAN with oral DSF is a more efficacious treatment of VISA infections compared to VAN monotherapy. To test this hypothesis, we will contrast the ability of these treatments to decrease morbidity and mortality in a VISA-induced peritonitis model. The pharmacokinetic parameters of DSF-VAN combinations and the effect of oral DSF on the intestinal microbiota will be determined as additional objectives of the in vivo studies. Collectively, we believe that the pharmacological and in vivo studies are poised to deliver novel discoveries about DSF as an adjunct therapy for treating infections and other diseases. The studies in this R15 REAP grant will further provide graduate and health professional students with diverse hands-on research opportunities as a training platform for careers in the biomedical and clinical sciences.